Background: Established in 1998, this R01 program has led the development of genome-scale reconstructions of bacterial metabolism, with special emphasis on E. coli. These reconstructions have been disseminated to the research community at large and led to the generation of many scientific studies from laboratories around the world. These genome-scale metabolic reconstructions are turning out to be a common denominator in the study of systems biology in microbes. Proposed Program: This continuation proposal outlines a program with two specific aims. The first aim is to continue to develop reconstruction technologies and expand the scope of the networks currently being reconstructed to include, on a genome-scale, metabolism, regulation of gene expression, including two- component signaling, as well as transcription and translation. The fulfillment of this aim will result in the largest and most comprehensive network reconstruction for any organism. The second aim is to use the metabolic reconstruction, which now comprehensively represents known metabolism in E. coli, for prospective discovery of new metabolic capabilities in E. coli. Such discovery is accomplished through a gap filling procedure that follows high-throughput phenotyping experiments of wild-type and knock-out strains. This second aim is novel and represents fulfillment of the promise of systems biology to systematically discover new biological functions through integrative computational and experimental studies. General Impact: This program, if renewed, will continue to pioneer the new genome-scale science of prokaryotic cells. It will lay the foundation for similar analysis of human pathogens, environmentally important organisms and those of bioterrorism importance. The results will thus have an impact on both basic and applied studies of bacteria.